TGF-β is a central regulator of chronic liver disease through induction of fibrogenic responses (Weiskirchen and Tacke (2016) Dig Dis 34:410-422; Katz et al. (2016) Cancer letters 379:166-172; Yoshida et al. (2014) Int J Oncol 45:1363-1371; Fabregat et al. (2016) The FEBS journal 283:2219-2232; Xu et al. (2016) J Histochem Cytochem 64:157-167). Although infiltrating macrophages are a source of TGF-β, hepatic stellate cells are a significant source of TGF-β in liver fibrosis. TGF-β stimulates induction of myofibroblast-like properties of hepatic stellate cells to produce extracellular matrix, leading to fibrosis. Although TGF-β inhibits hepatocyte proliferation under basal conditions, it has pro-oncogenic properties during malignant progression through stimulating epithelial to mesenchymal transition, cell survival and migration, and reduced immune surveillance.
TSP1 expression is increased in human liver disease with the THBS1 gene identified as part of the characteristic gene signature of chronic liver disease, including cirrhosis, in humans (Smalling et al. (2013) Am J Physiol Gastrointest Liver Physiol 305:G364-374). In vitro studies show that bile acids increase expression of TSP1 by hepatocytes, resulting in increased TGF-β signaling in co-cultured hepatic stellate cells (Myung et al. (2007) Biochem Biophys Res Commun 353:1091-1096). Both TSP1 and TGF-β are increased in congenital hepatic fibrosis (El-Youssef (1999) Journal of pediatric gastroenterology and nutrition 28:386-392). THBS1 message levels are increased in human liver specimens from patients with alcohol cirrhosis, NASH cirrhosis, and fibrosis and in mouse models of liver fibrosis induced by carbon tetrachloride or DDC (Smalling et al., (2013) Am J Physiol Gastrointet Liver Physiol 305: G364-G374.) TSP1 regulated TGF-β activation prevented hepatocyte proliferation and liver regeneration after partial hepatectomy in Thbs1 deficient mice (Hayashi et al. (2012) Hepatology 55:1562-1573) and TSP1 induction by obstructed portal flow in mice is thought to lead to TGF-β-dependent liver atrophy (Hayashi et al. (2016) Hepatol Res 55: 1562-1573). TSP1 has been shown to regulate latent TGF-β activation in animal models of liver fibrosis and in cell culture models (reviewed in Li et al. (2016) Hepatol Res, doi: 10.1111/hepr.12787). Treatment of rats with the TSP1 antagonist peptide LSKL prevented TGF-β activation and reduced liver fibrosis in the dimethylnitrosamine model (Kondou et al. (2003) J Hepatol 39:742-748). TSP1 is required for TGF-β signaling in both cultured hepatocytes and hepatic stellate cells, which is blocked by LSKL peptide (Breitkopf et al. (2005) Gut 54:673-681; Narmada (2013) J Cell Physiol 228:393-401). Interestingly, TSP1-dependent latent TGF-β activation might play a role in hepatitis C induced fibrosis and carcinogenesis as the hepatitis C core protein induces TSP1 expression by hepatocytes to increase active TGF-β and LSKL peptide blocks hepatitis C core protein activation of TGF-β (Benzoubir et al. (2013) J Hepatol 59:1160-1168). LSKL peptide administered early after injury also accelerated liver regeneration in mice following partial hepatectomy through blocking TGF-β activation and signaling (Kuroki et al. (2015) Br J Surg 102:813-825). Both the TGF-β1 and the TGF-β2 isoforms are upregulated in mouse models and in human tissues with liver fibrosis and also hepatocellular carcinoma (Dropmann et al. (2016) Oncotarget 7:19499-19518): this is interesting since TSP1 can activate both the β1 and β2 isoforms of latent TGF-β, whereas β2 cannot be activated by integrin-dependent mechanisms.
Genetic ablation of TGF-β, its receptors, or its signaling mediators results in developmental defects, inflammation, and increased carcinomas. Thus, it is therapeutically advantageous to target only adverse TGF-β activity in liver disease and spare homeostatic activity. Current anti-TGF-β therapeutics target the molecule itself or downstream signaling pathways and provide no mechanism for distinguishing between homeostatic and disease-related TGF-β activity, thereby increasing the potential for adverse effects. In fact, Smad 2 resistance and increased papilloma incidence in mice treated for 20 weeks with a TGF-β receptor kinase inhibitor have been identified (Connolly et al. (2011) Cancer Res 71:2339-2349) and the 1D11 pan-specific anti-TGF-β neutralizing antibody shows epithelial hyperplasia and progression to carcinoma in some models (Prud'homme (2007) Lab Invest 87:1077-1091).
TGF-β is secreted as a biologically inactive growth factor and control of the conversion of latent TGF-β to a biologically active growth factor is a major regulatory node. Binding of the N-terminal latency associated peptide (LAP) prevents TGF-β binding to its receptors and this interaction must be disrupted for TGF-β signaling to occur. Latent TGF-β can be converted to the active form through multiple mechanisms that include proteolysis, binding to integrins, mechanical forces, modifications of the latent complex by viral enzymes or by reactive oxygen species, or by binding to the secreted and ECM protein TSP1 (Sweetwyne and Murphy-Ullrich (2012) Matrix Biol 31:178-186; Murphy-Ullrich and Poczatek (2000) Cytokine Growth Factor Rev 11:59-69). The mechanism that regulates latent TGF-β activation can vary with tissue, cell type, and specific disease milieu. Blockade of the major activation mechanism in a particular disease typically attenuates adverse effects of TGF-β. Thus, it is important to identify the predominant mechanism of TGF-β activation in multiple myeloma.
Thrombospondin 1 (TSP1) is a complex multi-functional protein released from platelet α-granules, incorporated into the fibrin clot, and expressed by cell types that participate in wound healing responses in a temporally regulated manner (Agah et al. (2002) Am J Pathol 161:831-839; Murphy-Ullrich and Mosher (1985) Blood 66:1098-1104; DiPietro et al. (1996) Am J Pathol 148:1851-1860; Reed et al. (1993) J Histochem Cytochem 41:1467-1477; Raugi et al. (1987) J Invest Dermatol 89:551-554). TSP1 regulates multiple cellular events involved in tissue repair including hemostasis, cell adhesion, migration, proliferation, ECM expression and organization, and regulation of growth factor activity (Adams and Lawler (2004) Int J Biochem Cell Biol 36:961-968; Adams and Lawler (2011) Cold Spring Harb Perspect Biol 3:a009712). In addition to physiologic repair, TSP1 is also expressed at elevated levels in many tissues undergoing fibro-proliferative remodeling and blockade of specific actions of TSP1 or loss of TSP1 expression can attenuate pathologic tissue remodeling (Hugo (2003) Nephrol Dial Transplant 18:1241-1245; Poczatek et al. (2000) Am J Pathol 157:1353-1363; Daniel et al. (2007) Diabetes 56:2982-2989). TSP1 is a major regulator of latent TGF-β activation (Murphy-Ullrich and Poczatek (2000) Cytokine Growth Factor Rev 11:59-69). TSP1 also has TGF-β-independent functions in hemostasis, cell adhesion, migration, and growth factor regulation, e.g. regulation of epidermal growth factor (EGF), VEGF, and fibroblast growth factor (FGF) (Adams and Lawler (2011) Cold Spring Harb Perspect Biol 3:a009712). TSP1 is an endogenous angiogenesis inhibitor via inhibition of VEGF and FGF signaling. TSP1 binding to Cluster of Differentiation 47 (CD47) and Cluster of Differentiation 36 (CD36) blocks nitric oxide signaling.
TSP1 is a secreted ECM protein that controls TGF-β activity by binding and activating latent TGF-β (Sweetwyne and Murphy-Ullrich (2012) Matrix Biol 31:178-186; Murphy-Ullrich and Poczatek (2000) Cytokine Growth Factor Rev 11:59-69). TSP1 binds to latent TGF-β to activate TGF-β at the cell surface or in the extracellular milieu (Sweetwyne and Murphy-Ullrich (2012) Matrix Biol 31:178-186). Activation occurs through binding of the KRFK (-lysine-arginine-phenylalanine-lysine-) sequence in the TSP1 type 1 repeats (TSRs) to LSKL (-leucine-serine-lysine-leucine-) in the LAP of the latent complex, which disrupts LAP-mature domain interactions to expose the receptor binding sequences on the mature domain, rendering TGF-β capable of signaling (Young and Murphy-Ullrich (2004) J Biol Chem 279:38032-38039). Peptide mimetics of sequences involved in TSP1-TGF-β binding competitively inhibit TSP1-TGF-β activation and studies with these peptides have established TSP1 as a primary regulator of TGF-β bioactivity in different diseases (Sweetwyne and Murphy-Ullrich (2012) Matrix Biol 31:178-186). The tetrapeptide LSKL, which competitively blocks TSP-LAP binding, has been used in rodent models to inhibit TSP1-TGF-β activation and attenuate disease. Dose dependent intraperitoneal injection (i.p.) of LSKL improves end organ function in murine diabetic nephropathy and rat cardiomyopathy by blocking TGF-β signaling in target tissues (Belmadani et al. (2007) Am J Pathol 171:777-789; Lu et al. (2011) Am J Pathol 178:2573-2586). Animals necropsied after 15 weeks of treatment with 30 mg/kg i.p. LSKL, 3 times weekly, showed no inflammation, no tumors in all major organs, and no impairment of wound healing (Lu et al. (2011) Am J Pathol 178:2573-2586).
In vitro studies have shown that TSP1 activates latent TGF-β secreted by multiple cell types including endothelial cells, mesangial cells, hepatic stellate cells and skin, lung, and cardiac fibroblasts, T cells, and macrophages (Breitkopf et al. (2005) Gut 54:673-681; Murphy-Ullrich and Poczatek (2000) Cytokine Growth Factor Rev 11:59-69; et al. (2000) Am J Pathol 157:1353-1363; Mimura et al. (2005) Am J Pathol 166:1451-1463; Yehualaeshet et al. (1999) Am J Pathol 155:841-851; Zhou et al. (2006) Biochem Biophys Res Commun 339:633-641; Schultz-Cherry and Murphy-Ullrich (1993) J Cell Biol 122:923-932; Yevdokimova et al. (2001) J Am Soc Nephrol 12:703-712; Yang et al. (2009) J Autoimmun 32: 94-103; Zhou et al. (2004) Am J Pathol 165:659-669). Peptides such as LSKL or WxxW which block TSP1 binding to the latent complex or antibodies which block TSP1-dependent TGF-β activation such as monoclonal antibody 133 (Mab 133) have been used to establish the involvement of endogenous TSP1 in TGF-β activation in a number of disease conditions and physiologic processes (Belmadani et al. (2007) Am J Pathol 171:777-789; Lu et al. (2011) Am J Pathol 178:2573-2586; Crawford et al. (1998) Cell 93:1159-1170; Daniel et al. (2004) Kidney Int 65:459-468; Kondou et al. (2003) J Hepatol 39:742-748).
Initial evidence for an in vivo role of TSP1 in latent TGF-β activation was shown by the ability of the KRFK peptide administered in the perinatal period to partially rescue the abnormal TSP-1 null phenotype, in particular airway epithelial hyperplasia and pancreatic islet hyperplasia/acinar hypoplasia (Crawford et al. (1998) Cell 93:1159-1170). Furthermore, treatment of wild type mice with the LSKL blocking peptide in the perinatal period replicated features of the TSP1 knockout phenotype in the airways and pancreas. Double knockout of both β6 integrin and TSP1 results in a phenotype distinct from either single knockout that is characterized by severe inflammation, cardiac degeneration, and epithelial hyperplasia, suggesting both separate and synergistic roles in regulating latent TGF-β activation (Ludlow et al. (2005) J Cell Mol Med 9:421-437). However, it is likely that the primary role for TSP1 in controlling TGF-β activation is during injury, under stress, and in pathologic conditions, rather than during development. The expression of TSP1 is induced by factors associated with systemic diseases with fibrotic end organ involvement including high glucose, reactive oxygen species, and angiotensin II (Zhou et al. (2006) Biochem Biophys Res Commun 339:633-641; Yevdokimova et al. (2001) J Am Soc Nephrol 12:703-712; Wang et al. (2002) J Biol Chem 277:9880-9888; Wang et al. (2004) J Biol Chem 279:34311-34322). Indeed there is evidence from studies utilizing TSP1 antagonist peptides and diabetic TSP1 knockout mice that TSP1 is a major factor in the development of fibrotic end organ complications in diabetes (Daniel et al. (2007) Diabetes 56:2982-2989; Belmadani et al. (2007) Am J Pathol 171:777-789; Lu et al. (2011) Am J Pathol 178:2573-2586). Treatment with i.p. injections of LSKL, but not LSAL (leucine-serine-alanine-leucine) control peptide, reduced cardiac fibrosis, Smad phosphorylation, and improved left ventricular function (Belmadani et al. (2007) Am J Pathol 171:777-789). Similarly, treatment of Akita mice, a model of type 1 diabetes, with i.p. LSKL reduced urinary TGF-β activity and renal phospho-Smad 2/3 levels and improved markers of tubulointerstitial injury and podocyte function. (Lu et al. (2011) Am J Pathol 178:2573-2586). Both TSP1 and TGF-β are upregulated in pulmonary arterial hypertension due to chronic hypoxia, Schistosomiasis, and in scleroderma: recent studies show that TSP1 knockout or treatment with the blocking peptide LSKL protected against development of pulmonary hypertension due to hypoxia or Schistosome infection and also reduced active TGF-β (Kumar R et al, (2017) Nature Commun. 8: 15494). Epidermolysis bullosa is a disfiguring, blistering skin disease due to genetic defects in collagen and collagen anchoring fibrils that link the epidermis to the dermis. It has a fibrotic phenotype associated with increased TGF-β activity and thus TGF-β antagonists have been proposed as therapeutic agents (Nystroem A et al, (2015) EMBO Mol Medicine 7: 1211-1228). Losartan reduces TGF-β activity, inflammation, and the increased TSP-1 expression in a collagen VII hypomorphic model of epidermolysis bullosa (Nystroem A, et al). Interestingly, several studies have shown that TSP1 is involved in alveolar macrophage-dependent TGF-β activation in mouse and rat models of bleomycin-induced pulmonary fibrosis and treatment with either TSP1 or CD36 antagonist peptides can ameliorate lung fibrosis and reduce active TGF-β (Chen et al. (2009) Exp. Toxicol. Pathol. 61: 59-65; Yehualaeshet et al. (2000) Am. J. Respir. Cell Mol. Biol. 23: 204-12).
One of the roles of TSP1 in dermal wound healing appears to be regulating the activation of latent TGF-β. The phenotype of excisional wound healing in the TSP1 null mouse is consistent with a decrease in local TGF-β activation (Agah et al. (2002) Am J Pathol 161:831-839) and is characterized by a delay in macrophage recruitment and capillary angiogenesis and a persistence of granulation tissue, neovascularization, and inflammation (Nor et al. (2005) Oral Biosci Med 2:153-161). Topical treatment of TSP1 null wounds with the KRFK activating peptide largely rescued the TSP1 null wound phenotype (Nor et al. (2005) Oral Biosci Med 2:153-161). TGF-β levels in these wounds were increased following KRFK treatment and the effects of the KRFK peptide were blocked by a pan-specific anti-TGF-β antibody. While these data suggest that TSP1 plays a role in local activation of TGF-β during wounding, the studies of Agah et al., concluded that the decreased active and total TGF-β in the wounds of TSP1 or TSP1/TSP2 null mice is indirect and primarily due to defects in macrophage recruitment to wounds (a maj or source of TGF-β in wounds) leading to an overall reduction in TGF-β rather than a defect in activation (Agah et al. (2002) Am J Pathol 161:831-839). Despite this controversy, it is clear that TSP1 has the potential to modify the wound healing process. Subcutaneous implantation of TSP1 soaked sponges increased levels of active TGF-β, gel contraction and fibroblast migration (Sakai et al. (2003) J Dermatol Sci 31:99-109). Overexpression of TSP1 in keloids and in scleroderma correlates with increased TGF-β activity (Mimura et al. (2005) Am J Pathol 166:1451-1463; et al. (2000) Cell Death Differ 7:166-176; Chen et al. (2011) Fibrogenesis Tissue Repair 4:9). Others have used a derivative of the KRFK sequence, KFK (lysine-phenylalanine-lysine) coupled to a fatty acyl moiety to locally activate TGF-β and increase TIMP-1, which reduces MMP-induced elastin and collagen degradation when applied to dermal fibroblast cultures (Cauchard et al. (2004) Biochem Pharmacol 67:2013-2022). Systemic administration of the LSKL blocking peptide did not reduce Smad signaling or impair dermal wound healing in diabetic mice, although, these studies did not address the effects of direct LSKL administration to the wounds and it is not known if local dermal levels of LSKL following systemic intraperitoneal peptide administration are sufficient to alter local TGF-β activation (Lu et al. (2011) Am J Pathol 178:2573-2586).
Although peptides comprising the amino acid sequence LSKL capable of stimulating TGF-β activity are known, these peptides are often costly and difficult to synthesize. Moreover, small molecules such as LSKL have an extremely short plasma stability half-life, only 2.1 minutes. Thus, there remains a need for small molecules capable of altering TGF-β activity that are less expensive, easier to synthesize, and have an extended plasma stability half-life and methods of making and using same.